Week 5: Friday Flashcards
Typical ER signal sequence
Signal sequences typically include 6-15 hydrophobic residues preceded by an arginine (R) or lysine (K). Often near the N-terminus
Purpose of the positively charged amino acid in an ER signal sequence
The positively charged amino acid keeps the N-terminus in the cytosol
What removes the signal peptide for many proteins that are targeted into the ER?
The signal peptide is removed by signal peptidase (a proteolytic enzyme).
How are signal sequences inserted into the ER?
These signal sequences are inserted into the ER in a hairpin configuration (N-terminus stays on cytosolic side of the membrane)
When polysomes translate a cytosolic protein, where is the mRNA?
The mRNA encoding a cytosolic protein remains free in cytosol.
When polysomes translate a secretory protein with an N-terminal signal sequence, where is the mRNA?
mRNA encoding a protein targeted to the ER remains membrane-bound.
What three things does the signal sequence direct to the ER membrane?
- The ribosome
- mRNA
- The associated nascent chain
The translocon
A pore in the ER through which the signal sequence directs the growing polypeptide chain.
Co-translational translocation
The process by which a signal sequence directs the growing polypeptide chain into the lumen of the ER through the translocon. The signal sequence is then cleaved by signal peptidase in the lumen, making it a soluble component of the ER lumen.
SRP
Signal recognition particle (the signal sequence receptor)
What type of experiments were used to discover SRP?
Biochemical reconstitution experiments. They aim to test if a labeled protein was translocated into an organelle.
Composition of SRP
SRP is composed of an RNA molecule and 6 protein subunits.
Three steps of SRP’s function.
- Recognizes signal sequence as it emerges from ribosome.
2 .Causes a pause in translation. - Delivers ribosome with associated mRNA and partially translated protein to SRP receptor on ER membrane.
Function of SRP receptor
The SRP receptor serves as a docking platform and it facilitates handoff of signal sequence to the translocon.
What process controls the binding and release cycles of SRP?
The SRP receptor and SRP are both GTPases. The binding and release cycles are controlled by GTP hydrolysis.
How was the translocon discovered?
The translocon was discovered by a yeast genetic approach.
Why is a tight seal in the ER necessary during the translocation process?
The ER stores calcium, so a tight seal must be maintained during the process (Sec61 translocon) so it doesn’t escape.
State of translocon when not engaged
A central plug closes the translocon when it isn’t engaged
What happens to the translocon when the signal sequences binds to the receptor?
Binding of the signal sequence causes the plug to swing down and out of the channel. For many proteins, the signal sequence is cleaved off by the ER resident enzyme, signal peptidase.
Which type of proteins require a cleavable signal sequence?
Soluble proteins that are secreted, or sorted to the lumen of organelles in the secretory or endocytic pathways, require a cleavable signal sequence.
Structure of the translocon pore ring
The pore ring is made of the side chains of hydrophobic amino acids (mostly isoleucine). These side chains form a gasket around the polypeptide chain and brush away water and ions.
What are the three modes of protein translocation?
- Co-translational translocation (occurs in bacteria, archaea, and eukaryotes)
- Post-translational translocation (eukaryotes)
- Post-translational translocation (bacteria)
What is mutated in patients with autosomal-dominant polycystic liver disease?
Sec63
BiP is a member of what protein family?
BiP is a member of the Hsp70 family of ATPases that is localized to the ER lumen.
Interaction between Sec63 and BiP
Sec63 interacts with BiP to help recruit this Hsp70 protein to the translocating peptide.
SecA
An Hsp70 member protein required for translocation across the bacterial plasma membrane.
Steps to the reconstitution approach
- Break apart the cell
- Purify the components
- Put back together and see if the processes can be restored.
